Stem cells, unlike differentiated cells have the capacity to divide and either self-renew or differentiate into phenotypically and functionally different daughter cells (Keller, Genes Dev. 2005; 19:1129-1155; Wobus and Boheler, Physiol Rev. 2005; 85:635-678; Wiles, Methods in Enzymology. 1993; 225:900-918; Choi et al, Methods Mol Med. 2005; 105:359-368).
The pluripotency of mouse embryonic stem cells (ESCs) and their ability to differentiate into cells from all three germ layers makes embryonic stem cells an ideal source of cells for regenerative therapy for many diseases and tissue injuries (Keller, Genes Dev. 2005; 19:1129-1155; Wobus and Boheler, Physiol Rev. 2005; 85:635-678). However, this very property of embryonic stem cells also poses a unique challenge, i.e. generating the appropriate cell types for the treatment of a specific diseased or injured tissue in sufficient quantity and homogeneity to ensure therapeutic efficacy, and inhibiting the generation of other cell types that may have a deleterious effect on the tissue repair and regeneration. At present, protocols that either enhance differentiation of embryonic stem cells towards specific lineages and/or enrich for specific tissue cell types are too inefficient and generally yield heterogeneous cell populations that might be tumorigenic (Keller, Genes Dev. 2005; 19:1129-1155; Wobus and Boheler, Physiol Rev. 2005; 85:635-678).
Mesenchymal stein cells (MSCs) are multipotent stem cells that have documented evidence of therapeutic efficacy in treating musculoskeletal injuries, improving cardiiac function in cardiovascular disease and ameliorating the severity of GVHD (Le Blanc and Pittenger, 2005). Being lineage restricted, they have limited but robust potential to differentiate into mesenchymal cell types, e.g adipocytes, chondrocytes and osteocytes, and have negligible risk of teratoma formation. Host immune rejection of transplanted MSCs is routinely circumvented through autologous or allogeneic transplantation. MSCs can be isolated from several adult tissues including bone marrow (BM), adipose tissues (ad), cord blood and expanded ex vivo. However, availability of tissues for their isolation remains limiting and requires risky invasive procedures, and ex vivo expansion of MSCs while significant, is nonetheless finite.
An alternative source of MSCs is the unlimited supply of infinitely expandable and pluripotent human embryonic stem cells (hESCs) that will also eliminate the need for potentially risky invasive techniques. Host immune rejection of hESC-derived MSCs (hESC-MSC) could potentially be circumvented by either autologous hESCs generated by therapeutic cloning or immune compatible allogeneic hESCs when banks of hESC lines become sufficiently large.
The isolation of MSC or MSC-like cells from hESC has been previously described. Barberi, et al. (2005) PLoS Med 2, e161 describes a protocol which involves co-culturing hESCs with mouse OP9 cells in the presence of serum for 40 days before sorting for CD73+ cells that constitute about 5% of the total cell population. Xu, C. et al. (2004) Stem Cells 22, 972-80 describes a protocol which involves infecting hESC-derived embryoid bodies with a retrovirus expressing hTERT (Xu et al). However, the critical components of these protocols i.e. viral infection of exogenous DNA, exposure to mouse cells and use of serum introduce unacceptable risks of tumorigenicity and xenozootic infection, and preclude the use of these MSCs for clinical applications.
This invention seeks to solve this and other problems with methods in the art.